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Veterinary immunology and immunopathology2012; 146(2); 135-142; doi: 10.1016/j.vetimm.2012.02.006

Generation and characterization of monoclonal antibodies to equine CD16.

Abstract: The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the closely related Fc receptor CD32 expressed in the same system. In flow cytometric analysis with equine peripheral leukocytes, the mAbs labeled cells in the granulocyte, monocyte, and lymphocyte populations in a pattern consistent with other species. Monocytes that were strongly labeled with CD16 mAb 9G5 were also positive for the LPS receptor CD14. Cytospins made with peripheral leukocytes were immunohistochemically labeled and showed mAb recognition of primarily mononuclear cells. ELISA revealed that the nine mAbs can be grouped into three patterns of epitope recognition. These new antibodies will serve as useful tools in the investigation of equine immune responses and inflammatory processes.
Copyright © 2012 Elsevier B.V. All rights reserved.
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Publication Date: 2012-02-23 PubMed ID: 22424928PubMed Central: PMC3684017DOI: 10.1016/j.vetimm.2012.02.006Google Scholar: Lookup
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  • Extramural
  • Research Support
  • Non-U.S. Gov't
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  • U.S. Gov't
  • Non-P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research presents the generation and characterization of mouse monoclonal antibodies designed to recognize the low-affinity Fc receptor CD16 in horses. They offer valuable resources to investigate equine immune responses and inflammatory processes.

Objective and Methodology

  • The scientists embarked on this research due to the absence of studies around the expression and function of the CD16 receptor in horses. CD16 is a low-affinity Fc receptor that has a key role in numerous species’ inflammatory and innate immune responses.
  • To generate these antibodies, the team expressed the predicted extracellular region of equine CD16 as a recombinant fusion protein with equine interleukin-4 (rIL-4/CD16).
  • From this process, they were able to produce a range of mouse monoclonal antibodies (mAbs) that were capable of identifying equine CD16.

Characterization of Antibodies

  • Out of the set of generated antibodies, nine mAbs were selected for further characterization because they recognized CD16, but not interleukin-4, in enzyme linked immunosorbent assay (ELISA).
  • All nine mAbs were found to identify full-length, cell-surface CD16 when it was expressed as a green fluorescent protein (GFP) fusion protein by CHO (Chinese hamster ovary) cells.
  • Importantly, these mAbs did not recognize the closely related Fc receptor CD32 even when expressed in the same system, demonstrating their specificity to CD16.

Application and Surveillance

  • The monoclonal antibodies were also tested using flow cytometric analysis, demonstrating that they labeled cells in the granulocyte, monocyte, and lymphocyte populations in a manner comparable to other species.
  • Particularly, monocytes which were strongly marked with CD16 monoclonal antibody 9G5 were also positive for the lipopolysaccharide (LPS) receptor CD14.
  • Cytospin samples prepared with peripheral leukocytes were immunohistochemically labeled and predominantly demonstrated mAb recognition in mononuclear cells.
  • The ELISA studies revealed that these nine mAbs can be categorized into three patterns of epitope recognition.
  • Thus, the researchers conclude that these newly formed antibodies will be valuable tools for researching equine immune responses and inflammatory processes in the future.

Cite This Article

APA
Noronha LE, Harman RM, Wagner B, Antczak DF. (2012). Generation and characterization of monoclonal antibodies to equine CD16. Vet Immunol Immunopathol, 146(2), 135-142. https://doi.org/10.1016/j.vetimm.2012.02.006

Publication

Veterinary immunology and immunopathology
ISSN: 1873-2534
NlmUniqueID: 8002006
Country: Netherlands
Language: English
Volume: 146
Issue: 2
Pages: 135-142

Researcher Affiliations

Noronha, Leela E
  • Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Harman, Rebecca M
    Wagner, Bettina
      Antczak, Douglas F

        MeSH Terms

        • Animals
        • Antibodies, Monoclonal / genetics
        • Antibodies, Monoclonal / immunology
        • CHO Cells
        • Cloning, Molecular / methods
        • Cricetinae
        • Enzyme-Linked Immunosorbent Assay / veterinary
        • Female
        • Flow Cytometry / veterinary
        • Horses / blood
        • Horses / immunology
        • Immunity, Innate / immunology
        • Immunohistochemistry / veterinary
        • Leukocytes, Mononuclear / immunology
        • Mice
        • Mice, Inbred BALB C
        • Receptors, IgG / immunology
        • Recombinant Fusion Proteins / genetics
        • Recombinant Fusion Proteins / immunology

        Grant Funding

        • F32 HD055794 / NICHD NIH HHS
        • R01 HD049545 / NICHD NIH HHS

        Conflict of Interest Statement

        The authors report no conflict of interests.

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        Citations

        This article has been cited 10 times.
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        2. Gressler AE, Lübke S, Wagner B, Arnold C, Lohmann KL, Schnabel CL. Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences.. Front Immunol 2022;13:896255.
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        3. Farschtschi S, Mattes M, Pfaffl MW. Advantages and Challenges of Differential Immune Cell Count Determination in Blood and Milk for Monitoring the Health and Well-Being of Dairy Cows.. Vet Sci 2022 May 27;9(6).
          doi: 10.3390/vetsci9060255pubmed: 35737307google scholar: lookup
        4. Larson EM, Babasyan S, Wagner B. IgE-Binding Monocytes Have an Enhanced Ability to Produce IL-8 (CXCL8) in Animals with Naturally Occurring Allergy.. J Immunol 2021 May 15;206(10):2312-2321.
          doi: 10.4049/jimmunol.2001354pubmed: 33952617google scholar: lookup
        5. Patel RS, Tomlinson JE, Divers TJ, Van de Walle GR, Rosenberg BR. Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet(+) B cells.. BMC Biol 2021 Jan 22;19(1):13.
          doi: 10.1186/s12915-020-00947-5pubmed: 33482825google scholar: lookup
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          doi: 10.1371/journal.pone.0233537pubmed: 32442209google scholar: lookup
        7. Rzepecka A, Żmigrodzka M, Witkowska-Piłaszewicz O, Cywińska A, Winnicka A. CD4 and MHCII phenotypic variability of peripheral blood monocytes in dogs.. PLoS One 2019;14(7):e0219214.
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        10. Noronha LE, Harman RM, Wagner B, Antczak DF. Generation and characterization of monoclonal antibodies to equine NKp46.. Vet Immunol Immunopathol 2012 Jun 15;147(1-2):60-8.
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